Thrombin receptor binding peptides

ABSTRACT

Peptides having the general sequence A-B-C-D-E-F-Z wherein A is Ala, B is pFPhe, C is, for example, Arg, D is Cha, E is, for example, hArg, F is, for example, Tyr, and Z is, for example NH 2 . The peptides are useful for promoting wound healing and arresting bleeding in a patient.

This application is a 371 of PCT/US 94/06672 filed Jun. 14, 1994 whichwas a continuation of U.S. Ser. No. 08/077,850 filed Jun. 16, 1993 nowU.S. Pat. No. 5,457,177.

BACKGROUND OF THE INVENTION

Thrombin is a powerful factor in regulating the state of thecardiovascular system. It is clear that thrombin is an integralcomponent involved in the formation of blood clots by catalyzing theconversion of fibrinogen to fibrin, which is an integral part of mostclots. In addition, thrombin is known to act directly on cells in theblood and in the interior blood vessel wall, and specifically toactivate platelets to form clots. Thrombin-induced platelet activationis particularly important for arterial thrombus formation, a processthat causes myocardial infarction and some forms of unstable angina andstroke. In addition, thrombin promotes inflammation and other cellularactivities. Thrombin is chemotactic for monocytes, mitogenic forlymphocytes, and causes endothelial cells to express the neutrophiladhesive protein GMP-140 on their surfaces and inhibits the growth ofthese cells. Thrombin elicits platelet-derived growth factor from theendothelium and is a mitogen for mesenchymal cells.

Because thrombin is capable of direct activation of cells, it is assumedthat at least one thrombin receptor exists. However, it has not beenpossible to detect the presence of a thrombin receptor by traditionalbinding studies, since thrombin is capable of binding a large number ofsites present on cells which do not directly mediate the cellularresponses to thrombin, and thus the background levels of binding areprohibitively high.

The thrombin-binding proteins that have been identified do not seem tofunction as transduction molecules (Gronke, R. S. et al., J Biol Chem(1987) 262:3030-3036; Okamura, T., et al., J Biol Chem (1978) 253:3435).Modified thrombins that are physiologically inactive seem to bind toplatelets in the same way as thrombin itself. Thus, the binding sitesidentified by traditional binding studies may not be related tofunctional thrombin receptors. Also, since thrombin is a protease, ifthe receptor were proteolytically cleaved by its interaction withthrombin, the receptor's ability to bind tightly to thrombin would bedecreased. All of the foregoing factors suggest that traditional bindingstudies in an effort to identify and characterize a thrombin receptormight ultimately be unproductive.

While it has been assumed that a thrombin receptor exists, no directexperimental evidence exists from the studies conducted so far, whetherproteolytic cleavage by thrombin is involved in its receptor activation.When thrombin is treated with reagents which covalently modify andrender it proteolytically inactive, its ability to stimulate plateletsis abolished (Berndt, M. C., et al., "Platelets in Biology andPathology" (1981) Elsevier/North Holland Biomedical Press, pp. 43-74;Martin, B. M., et at., Biochemistry (1975) 14:1308-1314; Tollefsen, D.M., et at., J Biol Chem (1974) 249:2646-2651; Phillips, D. R., ThrombinDiath Haemorrh (1974) 32:207-215; Workman, E. F., et al., J. Biol Chem(1977) 252:7118-7123; Greco, N. J., et al., Blood (1990) 75:1983-1990).The modified forms of thrombin described in the reports above containbulky or charged moieties that occupy the active site and also obscureadditional regions of the surface of thrombin that bind substrate (Bode,W., et al., Embo J (1989) 8:3467-3475). Some of the chemically-modifiedthrombins do not, in fact, block thrombin-induced platelet activation.

Coughlin, et at., WO 9214750, describes cloning and sequencing of DNAencoding the cell surface receptor for thrombin, and recombinantproduction of the thrombin receptor at cell surfaces. Assay systems forthe detection of thrombin and the evaluation of thrombin agonists andantagonists are described.

SUMMARY OF THE INVENTION

Thrombin receptor radioligands of the present invention are useful forscreening for antagonists of thrombin receptor activation. Theradioligands are potent (<100nM) and therefore particularly useful inlarge volume screening of potential thrombin receptor antagonists.Antagonists of thrombin receptor activation are useful as antithromboticagents. Thus, the invention includes both thrombin receptor radioligandsdefined below and a thrombin receptor binding assay using theradioligands, for identifying thrombin receptor antagonists.

In addition to the high potency of these radioligands, the radioligandshave specific chemical sites for introduction of the radiolabel toproduce a radio receptor assay.

The radioligands are peptides having at least six amino acids andfalling within the following general structure:

    A-B -C-D-E-F-Z

wherein

A=Thr, Ser, Ala, or Gly;

B=Phe or halogenated Phe;

C, when F is Tyr(I) or Tyr(I₂),=Leu, Val, Cha, Nle, Phe, Arg, hArg, orLys;

C, when F is Tyr,=NH--CH(R)CO--,

wherein R=(CH₂)_(n) NHCO(CH₂)₂ --C₆ H₃ IO,

(CH₂)_(n) NHCO(CH₂)₂ --C₆ H₄ IO,

CH₂ --C₆ H₄ IO, or

CH₂ --C₆ H₃ I₂ O,

wherein n=2, 3, or 4;

D=Leu or Cha;

E=Arg, hArg, or Lys;

F=Tyr, Tyr(I), or Tyr(I₂); and ps Z=OH, OCH₃, or NRR',

wherein RR'=H, alkyl, aryl, or 1-15 amino acids.

The invention also includes a procedure for identifying thrombinreceptor antagonists among thrombin receptor antagonist candidates, anddetermining thrombin receptor antagonist activity using theabove-described radioligands.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes thrombin receptor radioligands which mimic thehuman thrombin receptor, and methods for determining thrombin receptorantagonist activity. The method comprises:

(a) isolating and washing human platelets or human platelet membranes,

(b) incubating a combination of platelets or platelet membranes with athrombin receptor radioligand and a candidate thrombin receptorantagonist,

(c) cooling the incubated combination by adding cold sodium chloride,

(d) filtering and washing the colled incubated combination, and

(e) measuring bound radioactivity.

Preferably, the thrombin receptor radioligand is selected from the groupof compounds consisting of:

    Ala--pFPhe--Arg--Cha--hArg--Tyr(I)--NH.sub.2,

    Ala--pFPhe--Arg--Cha--hArg--Tyr(I.sub.2)--NH.sub.2,

    Ala--pFPhe--hArg--Cha--hArg--Tyr(I)--NH.sub.2,

    Ala--pFPhe--hArg--Cha--hArg--Tyr(I.sub.2)--NH.sub.2, and ##STR1##

The nomenclature used to describe the peptide compounds of the inventionfollows the conventional practice where the N-terminal amino group isassumed to be to the left and the carboxy group to the right of eachamino acid residue in the peptide. In the formulas representing selectedspecific embodiments of the present invention, the amino- andcarboxy-terminal groups, although often not specifically shown, will beunderstood to be in the form they would assume at physiological pHvalues, unless other, vise specified. Thus, the N-terminal H⁺ ₂ andC-terminal O⁻ at physiological pH are understood to be present thoughnot necessarily specified and shown, either in specific examples or ingeneric formulas. Free functional groups on the side chains of the aminoacid residues can also be modified by amidation, acylation or othersubstitution, which can, for example, change the solubility of thecompounds without affecting their activity.

In the peptides shown, each residue, where appropriate, is representedby a one or three letter designation, corresponding to the trivial nameof the amino acid, in accordance with the following conventional list:

    ______________________________________                                               Amino Acid                                                                             Symbol                                                        ______________________________________                                               Alanine  Ala                                                                  Arginine Arg                                                                  Asparagine                                                                             Asn                                                                  Aspartic acid                                                                          Asp                                                                  Cysteine Cys                                                                  Glutamine                                                                              Gln                                                                  Glutamic acid                                                                          Glu                                                                  Glycine  Gly                                                                  Histidine                                                                              His                                                                  Isoleucine                                                                             Ile                                                                  Leucine  Leu                                                                  Lysine   Lys                                                                  Methionine                                                                             Met                                                                  Phenylalanine                                                                          Phe                                                                  Proline  Pro                                                                  Serine   Ser                                                                  Threonine                                                                              Thr                                                                  Tryptophan                                                                             Trp                                                                  Tyrosine Tyr                                                                  Valine   Val                                                           ______________________________________                                    

In the specific peptides shown in the present application, the L-form ofany amino acid residue having an optical isomer is intended unless theD-form is expressly indicated.

Amino acid residues can be generally subclassified into four majorsubclasses as follows:

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH.

Basic: The residue has a positive charge due to association with H ionat physiological pH and the residue is attracted by aqueous solution soas to seek the surface positions in the conformation of a peptide inwhich it is contained when the peptide is in aqueous medium atphysiological pH.

Neutral/nonpolar: The residues are not charged at physiological pH andthe residue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium. These residues are also designated"hydrophobic" herein.

Neutral/polar: The residues are not charged at physiological pH, but theresidue is attracted by aqueous solution so as to seek the outerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot, and there will be an attraction for or repulsion from an aqueousmedium to a greater or lesser extent. To fit the definition of"charged," a significant percentage (at least approximately 25%) of theindividual molecules are charged at physiological pH. The degree ofattraction or repulsion required for classification as polar or nonpolaris arbitrary and, therefore, amino acids specifically contemplated bythe invention have been classified as one or the other. Most amino acidsnot specifically named can be classified on the basis of known behavior.

Amino acid residues can be further subclassified as cyclic or noncyclic,and aromatic or nonaromatic, self-explanatory classifications withrespect to the side chain substituent groups of the residues, and assmall or large. The residue is considered small if it contains a totalof 4 carbon atoms or less, inclusive of the carboxyl carbon. Smallresidues are, of course, always nonaromatic.

For the naturally occurring protein amino acids, subclassificationaccording to the foregoing scheme is as follows:

Acidic: Aspartic acid and Glutamic acid;

Basic/noncyclic: Arginine, Lysine;

Basic/cyclic: Histidine;

Neutral/polar/small: Glycine, serine, cysteine;

Neutral/nonpolar/small: Alanine;

Neutral/polar/large/nonaromatic: Threonine, Asparagine, Glutamine;

Neutral/polar/large aromatic: Tyrosine;

Neutral/nonpolar/large nonaromatic: Valine, Isoleucine, Leucine,Methionine;

Neutral/nonpolar/large/aromatic: Phenylalanine, and Tryptophan.

The secondary amino acid proline, although technically within the groupneutral/nonpolar/large/cyclic and nonaromatic, is a special case due toits known effects on the secondary conformation of peptide chains, andis not, therefore, included in this defined group.

Non-natural, uncommon amino acids, include, for example, beta-alanine(beta-Ala), or other omega-amino acids, such as 3-amino propionic,4-amino butyric and so forth, alpha-aminisobutyric acid (Aib), sarcosine(Sar), ornithine (Ore), citrulline (Cit), t,butylalanine (t-BuA),t-butylglycine (t-BuG), N-methylisoleucine (N-Melle), phenylglycine(Phg), and cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya)2-naphthylalanine (2-Nal); 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (Tic); β-2-thienylalanine (Thi); and methionine sulfoxide (MSO).These also fall conveniently into particular categories.

Based on the above definitions,

Sar and beta-Ala and Aib are neutral/nonpolar/small;

t-BuA, t-BuG, N-Melle, Nle, Mvl and Cha areneutral/nonpolar/large/nonaromatic;

Orn is basic/noncyclic;

Cya is acidic;

Cit, Acetyl Lys, and MSO are neutral/polar/large/nonaromatic; and

Phg, Nal, Thi and Tic are neutral/nonpolar/large/aromatic.

The various omega-amino acids are classified according to size asneutral/nonpolar/small (beta-Ala, i.e., 3-aminopropionic,4-aminobutyric) or large (all others).

Other amino acid substitutions can also be included in peptide compoundswithin the scope of the invention and can be classified within thisgeneral scheme according to their structure.

All of the compounds of the invention, when an amino acid forms theC-terminus, may be in the form of the pharmaceutically acceptable saltsor esters. Salts may be, for example, Na⁺, K⁺, Ca⁺², Mg⁺² and the like;the esters are generally those of alcohols of 1-6C.

Assays

Experiments were carded out to screen for the potency of thrombinreceptor-derived peptides and to identify receptor antagonists.

Assay to determine EC₅₀ values for peptides

Human platelets are isolated and washed as described in Connolly et al.(1992) J. Biol. Chem. 267, 6893-6898 and suspended at 2×10⁸ platelets/mlin a modified Tyrode's buffer without Ca⁺⁺. Platelets are incubated with0.2 mg/ml human fibrinogen for 2 minutes at 37° C. The sample istransferred to an aggregometer, stirred, the test compound is added andaggregation monitored for 5 min. The extent and rate of aggregation iscalculated by the Agglink Software program supplied by Chronolog Corp.EC₅₀ values are visually determined from plots of the extent and rate ofaggregation versus log concentration of test compound. The mean of thesevalues from 3 separate experiments performed on blood from differentdonors on different days is reported in μM.

Assay used to determine antagonist activity

Radioligand Binding Assay--(A) Preparation of radioligand Unlabeledpeptide is iodinated using the IODO-GEN radioiodination reagent togetherwith ¹²⁵ I!NaI. The non iodinated, mono and diiodinated species areseparated by C₁₈ reverse phase HPLC chromatography. The final specificactivities were between 60 and 570 Ci/mmole. (B) Human platelets areisolated and washed as above. Platelets are incubated with radioligand¹²⁵ I!-peptide alone or in the presence of thrombin receptor antagonistin a final volume of 250-500 μl for 10-60 min at 4°, 22° or 37° C. Thebinding assay is terminated by the addition of ice cold 0.15M NaClfollowed by filtration on Whatman GF/C filters that had been presoakedin 0.3% polyethylenimine. The filters are washed 3 more times with icecold buffer and the bound radioactivity on the filters monitored.Antagonist activity is noted as a decrease in counts bound as comparedto the control radioligand bound. (C) Human platelet membranes preparedaccording to Biochim. et Biophys Act (1986) 854, 67-76, made up in 50 mMTris HCL, pH 7.6, 1 mM EGTA, 10 mM MgCl₂, 62-250 μg assay, are incubatedas with platelets above. The remainder of the procedure is as with theplatelets.

Preferred radioligands of the present invention are hexapeptides havingEC₅₀ (μM) values<0.1 μM. EC₅₀ values, expressed in micromolar units,represent the concentration of measured compound required to elicit a50% maximal aggregation according to the platelet aggregation assay.These radioligands are particularly useful for quickly identifyingthrombin receptor antagonists.

The thrombin receptor ligands mimic the activated form of the thrombinreceptor protein and are useful in encouraging platelet aggregateformation in localized application. They also stimulate fibroblastproliferation and thus may be useful in promoting wound healing.

Specifically preferred radioligands, referenced by Sequence ID Number,are the following hexapeptides:

    ______________________________________                                                              EC.sub.50                                                                     (μM)                                                                            SEQUENCE                                           ______________________________________                                        AlapFPheArgChahArgTyr(I)NH.sub.2                                                                      0.030  1                                              AlapFPheArgChahArgTyr(I.sub.2)NH.sub.2                                                                0.150  2                                              AlapFPhehArgChahArgTyr(I)NH.sub.2                                                                     0.025  3                                              AlapFPhehArgChahArgTyr(I.sub.2)NH.sub.2                                                               0.090  4                                               ##STR2##               --     5                                              ______________________________________                                    

wherein pFPhe is para-fluorophenylalanine, hArg is homoarginine, Cha iscyclohexylalanine, I is iodine, Tyr(I) is monoiodinated tyrosine, andTyr(I₂) is diiodinated tyrosine.

Specifically preferred assays for identifying thrombin receptorantagonists are those described below which use the specificallypreferred radioligands.

Ligand Preparation

The nonradioactive radioligands of the present invention are prepared bystandard solid phase methodology (Merrifield, J. Am. Chem. Soc., 85,2149 (1964) and Stewart and Young, "Solid Phase Peptide Synthesis",Pierce Chemical Company, Rockford, Ill. (1984)), starting with amethylbenzhydrylamine resin (MBHR). The resin, R, is acylated with aprotected amino acid (X') and the α-amino group is deprotected,neutralized and acylated with the next amino acid in the desiredsequence. In this manner, the desired peptide-resin

    A'-B'-C-D'-E'-F-MBHR

is built. The peptide is cleaved from the solid support using HF-anisole(9:1) and the crude product is isolated by preparative HPLC.Radiolabeled amino acids are treated, e.g. with an iodinating reagent,prior to solid phase synthesis. In general, the procedure for preparingradioligands is represented by the following scheme: ##STR3## DMF isdimethylformamide, and HF is hydrogen fluoride.

EXAMPLE 1 Method for identifying thrombin receptor antagonists

Unlabeled peptide ##STR4## prepared according to the proceduresgenerally outlined above, is iodinated using the IODO-GENradioiodination reagent together with ¹²⁵ I!NaI, to form ##STR5## Thenon iodinated, mono and diiodinated species are separated by C₁₈ reversephase HPLC chromatography. The final specific activities were between 60and 570 Ci/mmole.

Human platelets are isolated and washed as above. Platelets areincubated with radioligand ¹²⁵ I!-peptide alone or in the presence ofthrombin receptor antagonist in a final volume of 500 μl for 30 min at22° C. The binding assay is terminated by the addition of ice cold 0.15MNaCl followed by filtration on Whatman GF/C filters that had beenpresoaked in 0.3% polyethylenimine. The filters are washed 3 more timeswith ice cold buffer and the bound radioactivity on the filtersmonitored. Antagonist activity is noted as a decrease in counts bound ascompared to the control radioligand bound.

EXAMPLE 2 Method for identifying thrombin receptor antagonists

Unlabeled peptide

    Ala--pFPhe--hArg--Cha--hArg--Tyr--NH.sub.2

prepared according to the procedures generally outlined above, isiodinated using the IODO-GEN radioiodination reagent together with ¹²⁵I!NaI, to form

    Ala--pFPhe--hArg--Cha--hArg--Tyr(I)--NH.sub.2.

The non iodinated, mono and diiodinated species are separated by C₁₈reverse phase HPLC chromatography. The final specific activities werebetween 60 and 570 Ci/mmole.

Human platelets are isolated and washed as above. Platelets areincubated with radioligand ¹²⁵ I!-peptide alone or in the presence ofthrombin receptor antagonist in a final volume of 500 μl for 30 min at22° C. The binding assay is terminated by the addition of ice cold 0.15M NaCl followed by filtration on Whatman GF/C filters that had beenpresoaked in 0.3% polyethylenimine. The filters are washed 3 more timeswith ice cold buffer and the bound radioactivity on the filtersmonitored. Antagonist activity is noted as a decrease in counts bound ascompared to the control radioligand bound.

Thrombin receptor antagonists--therapeutic uses

The thrombin receptor antagonists identified using the assay describedabove can be administered in such oral forms as tablets, capsules (eachof which includes sustained release or timed release formulations),pills, powders, granules, elixers, tinctures, suspensions, syrups, andemulsions. Likewise, they may be administered in intravenous (bolus orinfusion), intraperitoneal, subcutaneous, or intramuscular form, allusing forms well known to those of ordinary skill in the pharmaceuticalarts. An effective but non-toxic amount of the compound desired can beemployed as an anti-aggregation agent.

The thrombin receptor antagonists may be administered to patients whereprevention of thrombosis by thrombin inhibition is desired. They areuseful in surgery on peripheral arteries (arterial grafts, carotidendarterectomy) and in cardiovascular surgery where manipulation ofarteries and organs, and/or the interaction of platelets with artificialsurfaces, leads to platelet aggregation and consumption. The aggregatedplatelets may form thrombi and thromboemboli. They may be administeredto these surgical patients to prevent the formation of thrombi andthromboemboli.

Extracorporeal circulation is routinely used for cardiovascular surgeryin order to oxygenate blood. Platelets adhere to surfaces of theextracorporeal circuit. Adhesion is dependent on the interaction betweenGPIIb/IIIa on the platelet membranes and fibrinogen adsorbed to thesurface of the circuit. (Gluszko et al., Amer. J. Physiol.,252(H),615-621 (1987)). Platelets released from artificial surfaces showimpaired hemostatic function. The thrombin receptor antagonists may beadministered to prevent adhesion.

Other applications of the thrombin receptor antagonists includeprevention of platelet thrombosis, thromboembolism and reocclusionduring and after thrombolytic therapy and prevention of plateletthrombosis, thromboembolism and reocclusion after angioplasty orcoronary and other arteries and after coronary artery bypass procedures.They may also be used to prevent myocardial infarction and unstableangina.

The dosage regimen utilizing the thrombin receptor antagonists isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the patient; and the particularcompound or salt thereof employed. An ordinarily skilled physician orveterinarian can readily determine and prescribe the effective amount ofthe drug required to prevent, counter, or arrest the progress of thecondition.

Oral dosages of the thrombin receptor antagonists, when used for theindicated effects, will range between about 0.01 mg per kg of bodyweight per day (mg/kg/day) to about 100 mg/kg/day and preferably 1.0-100mg/kg/day and most preferably 1-20 mg/kg/day. Intravenously, the mostpreferred doses will range from about 1 to about 10 mg/kg/minute duringa constant rate infusion. Advantageously, the thrombin receptorantagonists may be administered in divided doses of two, three, or fourtimes daily. Furthermore, they can be administered in intranasal formvia topical use of suitable intranasal vehicles, or via transdermalroutes, using those forms of transdermal skin patches well known tothose of ordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will, or course,be continuous rather than intermittent throughout the dosage regime.

The thrombin receptor antagonists are typically administered as activeingredients in admixture with suitable pharmaceutical diluents,excipients or carriers (collectively referred to herein as "carrier"materials) suitably selected with respect to the intended form ofadministration, that is, oral tablets, capsules, elixers, syrups and thelike, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as lactose,starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the oral drug components can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water and the like. Moreover, whendesired or necessary, suitable binders, lubricants, distintegratingagents and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor beta-lactose, corn-sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsinclude, without limitation, starch methyl cellulose, agar, bentonite,xanthan gum and the like.

The thrombin receptor antagonists can also be administered in the formof liposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

The thrombin receptor antagonists may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The thrombin receptor antagonists may also becoupled with soluble polymers as targetable drug carriers. Such polymerscan include polyvinlypyrrolidone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the thrombin receptorantagonists may be coupled to a class of biodegradable polymers usefulin achieving controlled release of a drug, for example, polylactic acid,polyglycolic acid, copolymers of polylactic and polyglycolic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanoacrylates and cross linked oramphipathic block copolymers of hydrogels.

The thrombin receptor antagonists can also be co-administered withsuitable anti-coagulation agents or thrombolytic agents such asplasminogen activators or streptokinase to achieve synergistic effectsin the treatment of various ascular pathologies. They may also becombined with heparin, aspirin, or warfarin.

The thrombin receptor antagonists may be used for inhibiting integrinprotein-complex function relating to cell attachment activity. They maybe administered to patients where inhibition of human or mammalianplatelet aggregation or adhesion is desired.

The thrombin receptor antagonists are eliminated from circulationrapidly and are particularly useful in inhibiting platelet aggregationin situations where a strong antithrombotic of short duration oreffectiveness is needed. Thus, they may find utility in surgery onperipheral arteries (arterial grafts, carotid endaterectomy) and incardiovascular surgery where manipulation of arteries and organs, and/orthe interation of platelets with artificial surfaces, leads to plateletaggregation and consumption. The aggregated platelets may form thrombiand thromboemboli. The thrombin receptor antagonists may be administeredto these surgical patients to prevent the formation of thrombi andthromboemboli.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaXaaArgXaaXaaXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaXaaArgXaaXaaXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaXaaXaaXaaXaaXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaXaaXaaXaaXaaXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AlaXaaXaaXaaXaaTyr                                                            15                                                                            __________________________________________________________________________

What is claimed is:
 1. A peptide of the following general structure:

    A-B-C-D-E-F-Z

wherein A=Ala; B=pFPhe; C=Arg, hArg, or Lys; D=Cha; E=Arg or hArg; F=Tyror a bond; and Z=OH, OCH₃, or NH₂.
 2. A peptide of claim 1 selected fromthe group consisting of

    Ala--pFPhe--Arg--Cha--hArg--Tyr--NH.sub.2,

    Ala--pFPhe--hArg--Cha--hArg--Tyr--NH.sub.2.


3. A peptide of claim 2 which is Ala--pFPhe--Arg--Cha--hArg--Tyr--NH₂.4. A peptide of claim 2 which is Ala--pFPhe--hArg--Cha--hArg--Tyr--NH₂.